Contour mapping of spectral diagnostics

ABSTRACT

The present invention relates to a method of generating and processing spectral information arising from the laser induced fluorescence of tissue. The intensity of fluorescence is recorded as a function of both excitation and emission wavelengths and contour maps of tissue fluorescence are generated which are useful in the diagnosis of condition of the tissue under examination.

This is a continuation of co-pending application Ser. No. 07/342,311filed on Apr. 24, 1989, now abandoned.

BACKGROUND

The present invention relates to a system and method for processingspectral information used to aid in the diagnosis of diseased tissue.More particularly, it relates to a method for collecting and displayingthe excitation and emission spectra resulting from thelaser-induced-fluorescence of tissue.

Methods utilizing the laser-induced-fluorescence ("LIF") of tissue havebeen developed which permit the characterization of the tissue beingexamined. This technique has traditionally employed the use offluorescing agents or dyes which are introduced or applied to the tissueof interest which is then irradiated to induce fluorescence and producea spectrum that can be used to distinguish diseased from normal tissue.The emission spectra obtained through these methods are normally plottedas intensity versus emission wavelength for a given excitationwavelength. These graphical displays can provide information regardingthe diseased condition of tissue as specific peaks in the spectra ofabnormal tissue appear which are not present in normal tissue.

Laser catheter systems have also been developed, often in conjunctionwith known techniques for using fluorescence to aid in diagnosis, forthe purpose of inserting a light transmitting device into the human bodywhere laser radiation can be directed onto tissue adjacent the distalend of the catheter to induce fluorescence. The light emitted by thetissue is transmitted along the catheter and analyzed at the proximalend thereof to produce an emission spectrum of the irradiated tissue.

The problem with these standard spectra is that they are highlydependent upon the excitation wavelength that is used such that a highlyfragmented view of the spectral characteristics of the tissue isprovided. Thus, a more complete method of generating diagnosticinformation utilizing LIF spectroscopy is needed that more fullycharacterizes the spectral features of tissue.

SUMMARY OF THE INVENTION

The present invention relates to a system for characterizing tissuefluorescence in the ultraviolet and throughout the visible for thepurpose of generating diagnostically useful information. The presentsystem provides the ability to look at fluorescence over a wide range ofexcitation and emission wavelengths. Such a broad survey is important infully characterizing the spectroscopic differences between tissue typesas well as in aiding in the identification of tissue fluorophores whosepresence is correlated with known normal and abnormal states of tissue.

The intensity of fluorescence depends on three parameters: the radiativeproperties of the fluorophores within the tissue; the wavelength of theexciting light; and the wavelength of the emitted fluorescence. Twotypes of scans are typically used when recording fluorescence intensity:emission scans (spectra), in which the excitation wavelength is heldconstant and the emission wavelength is varied, and excitation scans(spectra), in which the emission wavelength is constant and theexcitation is varied. Either type of scan alone contains usefulinformation about the tissue constituents; however, both excitation andemission profiles are needed for a full characterization offluorescence. In fact, a complete fluorescence characterization involvesrecording the fluorescence intensity for each possible pair ofexcitation and emission wavelengths.

Typically, emission (or excitation) scans are presented as twodimensional plots with emission (or excitation) wavelengths runningalong one axis and fluorescence intensity running along the other axis.In order to display intensity for each possible pair of excitation andemission wavelengths, a 3-dimensional plot of intensity vs. excitationand emission is one alternative for displaying the information foranalysis. There are also two methods employed for representing suchplots in two dimensions: perspective drawing and contour (topographic)mapping. In a preferred embodiment, contour maps are employed to displaythe data as they provide for viewing many of the important features onone plot.

This method is employed in conjunction with laser catheter systems usedto induce fluorescence in bodily tissue both in vitro and in vivowithout the use of fluorescence enhancing agents, also referred toherein as "autofluorescence". The procedure is illustrated using datacollected on excised tissue samples, including artery wall,gastrointestinal tissue and bladder tissue, but are usefully applied toall types of tissue and the abnormal or diseased conditions which can bedifferentiated by optical characteristics.

In particular, fluorophores present in the tissue under study, whoseconcentration varies with the condition of the tissue, have beenassociated with specific features of the contour spectra. Thiscorrelation between the contour spectra and the chemical constituents ofthe tissue is useful in the construction of diagnostic methods which cansystematically differentiate tissue type and condition.

The above and other features of the invention, including various noveldetails in the methods described and certain combinations thereof, willnow be more particularly described with reference to the accompanyingdrawings and pointed out in the claims. It will be understood that theparticular methods of diagnosis embodying the invention are shown by wayof illustration only and not as a limitation of the invention. Theprinciple features of the invention may be employed in variousembodiments without departing from the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an apparatus used in conjunction withthe present invention.

FIG. 2 illustrates a contour map of morphologically normal aorta.

FIG. 3a presents four emission scans from the region about line A-B ofFIG. 2.

FIG. 3b presents emission scans from line A-C in FIG. 2.

FIGS. 4a and 4b present a contour map and a perspective view of anexcitation emission matrix of fluorescence intensity of normal aorta.

FIGS. 5a and 5b show a contour map and a perspective view, respectively,of an averaged excitation-emission matrix of fibrous aortic tissue.

FIGS. 6a and 6b show a contour map and a perspective view, respectively,of an averaged excitation-emission matrix of calcified aortic tissue.

FIGS. 7a and 7b show a contour map and a perspective view, respectively,of an excitation-emission matrix of fatty aortic tissue.

FIGS. 8a and 8b show an absorption spectrum and a contour map,respectively, of hemoglobin.

FIGS. 9a and 9b show contour maps of bulk aorta before and after thesample is soaked in saline.

FIG. 10 shows a contour map of the attenuation matrix of hemoglobin.

FIGS. 11a and 11b show contour maps of aortic tissue after soaking toremove hemoglobin, and after the addition of the effects of hemoglobinusing the hemoglobin absorption matrix.

FIG. 12 illustrates a contour 12 of powdered type 1 bovine collagen.

FIGS. 13a and 13b show average fluorescence contour maps of normal humancolon tissue.

FIGS. 14a and 14b show average fluorescence contour maps for adenomatoushuman colon tissue.

FIG. 15 shows the ratio of an average contour map for adenomatous polypto an average contour map for normal gastrointestinal tissue.

FIG. 16 shows the map of FIG. 15 where the contour line density isincreased by a factor of two to show more detail.

FIG. 17 illustrates a diagnostic algorithm for differentiatingadenomatous from normal colon tissue based upon a ratio of maps at anexcitation wavelength of 330 nanometers and an emission wavelength of385 nanometers.

FIG. 18 illustrates a diagnostic algorithm based upon an excitationwavelength of 440 nanometers and an emission wavelength of 480nanometers.

FIG. 19 illustrates a binary type diagnostic algorithm.

FIGS. 20a and 20b show average contour maps on different scales ofnormal bladder tissue.

FIGS. 21a and 21b show average contour maps on two different scales ofbladder tumor.

FIG. 22 shows a ratio of contour maps for bladder tumor and normalbladder tissue.

FIG. 23 shows the map of FIG. 22 with a greater contour line density.

FIG. 24 shows a binary diagnostic algorithm to differentiate bladdertumor from normal bladder tissue.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates a preferred embodiment of the system that is used ingenerating and processing matrices of fluorescence intensity obtained asa function of both excitation and emission wavelengths. Light source 10can be a broadband source such as a Xenon arc lamp. However, a tunablelaser can also be employed to provide excitation radiation of variablewavelength. A double monochromator 12 subject to scan control 14 directsradiation of selected wavelength through optical fiber 16 onto thetissue sample 22. Collection fibers 18 collect the fluorescent radiationemitted by the sample 22 and direct it through collection optics 20 andinto a second double monochromator 24. Photomultiplier 26 then detectsthe radiation and generates a signal that is forwarded to computer 28for processing and display. Computer 28, via line 30, can be used tocontrol the scanning parameters of the two monochromators 12 and 24.

The optical probe or catheter 32 can also be used in vivo within thehuman body to perform diagnostic scans on living tissue. The methods ofthe present invention may be used in conjunction with the laser cathetersystems disclosed in U.S. Pat. No. 4,718,417, incorporated herein byreference.

Fluorescence intensity from normal aorta was recorded over a range offrom 250 to 700 nm every 5 nm for both excitation and emission. Thesystem used for this type of measurement is easily tuned over a largerange of excitations. The system can use a rhodamine solution as areference, so that differences in intensity of the exciting light atdifferent wavelengths are automatically removed from the spectra. Thismakes it possible to obtain fluorescence intensities over a large rangeof excitations and emissions on a single sample of tissue in areasonable amount of time. Gratings blazed at 250 nm have been used sothat emission spectra can be taken from 250 to 700 nm.

To generate the data, emission scans were taken with excitations of 250to 500 nm every 5 nm. The emission ranges ran from the excitation +12.5nm to 697.5 nm every 5 nm. The FIGURES described below are generated bysequential acquisition of emission scans; however, excitation scanscould be used as well to display the relationship between excitation andemission. The FWHM (full width-half maximum) of the excitation sourcewas 2 nm, and the emission FWHM was 3 nm. An integration time of 1second at each point yielded a reasonable signal to noise ratio for allscans.

FIG. 2 shows the contour map that was generated from a sample ofmorphologically normal aorta. The map shows five distinct peaks (labeledA-E in FIG. 2). Each peak potentially represents a distinct fluorophore;however, it is known that fluorescence reabsorption in the tissue cancreate artificial peaks in emission spectra. On the contour map, suchreabsorption valleys should appear as depressions at constant emissionwavelength, since the wavelength of reabsorption does not depend onexcitation. The three dashed vertical lines in FIG. 2 are examples ofsuch reabsorption. The two lines near 570 nm emission representabsorption due to oxy-hemoglobin, while the line near 415 nm is theSoret band of heme. Based on this interpretation, the two peaks D and Eare due to a single fluorophore which is known to be elastin/collagen.The peaks B and C may also be due to a single fluorophore, although thefact that they are slightly shifted in excitation from one anothersupports the notion that the peaks are due to two distinct fluorophores.

Horizontal lines, that is, lines of constant excitation, on the maprepresent emission scans. Four emission scans from the region designatedby line A-B in FIG. 2 are shown in FIG. 3a. These scans are very similarto those with 476 nm excitation. The two peaks at 560 and 600 nm areartificial in that they are created by the reabsorption valleys at 540and 580 nm, as was surmised from the contour map.

Emission scans from line A-C are shown in FIG. 3b. As can be seen fromthe contour map, the intensity of fluorophore A, emitting at 340 nm, isstrongly dependent on excitation wavelength near 300 nm. This is videntfrom the very close spacing of the contour lines in this region. Thisfluorophore has been identified as tryptophan. The Soret valley at 415nm is also seen in these emission scans, as is the emergence of anotherpeak (B) at 390 nm, as excitation wavelength is made longer. Based onspectra from thin samples of artery, peak C (at 450 nm emission) wasassumed to be artificially created by the Soret valley, so that peaks Band C were assigned to a single fluorophore. However, as the contour mapreveals, these peaks occur at slightly different excitations, and it ispossible that two distinct fluorophores are being observed.

This system has been used to determine optimal excitation and emissionwavelengths for differentiating normal and atherosclerotic aorta. Also,contour maps of fibrous, fatty and calcified plaques have beenconstructed. By normalizing these maps and then subtracting them from,or ratioing them, to a normalized map of normal aorta, excitation andemission wavelengths at which fluorescence lineshapes of normal anddiseased tissue are most different can be easily identified.

FIGS. 4-7 present data collected as excitation-emission matrices (EEM's)of bulk samples of normal and pathological human aorta tissue. Thisdata, which is presented in the form of contour maps as well asperspective drawings is used to determine an optimum wavelength fordistinguishing between normal and pathological tissue.

Tissue samples are classified as either normal, fibrous, fatty, orcalcified. In all, there were nine normal samples run from fivedifferent patients six calcified samples which were all from the samepatient, eight fibrous samples taken from four different patients, andtwo samples of fatty tissue from the same patient.

The EEM's of each tissue type were averaged with the exception of thefatty sample shown in FIG. 7

The main features of the EEM's of the normal samples of FIGS. 4a and 4bare the dominant tryptophan peak in the uv and the absorption valleys at415 nm, which are believed to be caused by the presence of hemoglobin inthe tissue. There is also weaker fluorescence in the visible range,which is primarily due to chromophores within the collagen and elastin.The most noticeable difference between the EEM's of the normal andfibrous tissue (at FIGS. 5a and 5b) is that there is greaterfluorescence at the longer wavelengths in normal compared to fibrous.There also appears to be a difference in the location of the peak of thefluorescence due to chromophores within the collagen, but this may bethe result of less hemoglobin absorption in the fibrous tissue. Onedifference between the calcified tissue shown in FIGS. 6a and 6b and thenormal tissue is that the fluorescence peak due to tryptophan is about afactor of two lower in the calcified tissue. In addition, thefluorescence due to chromophores within the collagen and elastin is twoto three times greater in the calcified tissue, and there is also lessabsorption at 415 nm. One of the chromophores found within the collagenor elastin morphologic structures is pyridinoline which is known tofluoresce in the range 400-420 nm when excited by radiation in the rangeof 360-370 nm.

Hemoglobin (Hb) plays an important role in in vitro aorta EEM's. Thelarge Soret absorption band at 416 nm is responsible for the deepvalleys seen in the contour map both at excitations and emissions around416 nm. Oxyhemoglobin also produces the minor valleys near 540 and 580nm. Note that heme is one of the chromophores found in hemoglobin. Hemehas an absorption region centered around 560 nm. This conclusion issupported by the following observations:

1) The absorption spectrum of hemoglobin has peaks coincident with thevalleys mentioned above (FIGS. 8a and 8b).

2) In vitro aorta samples have typically been exposed to lysed red bloodcells and, therefore, free Hb which can readily diffuse into the tissue.

3) These valleys are absent in 10 μm sections of intima and media. Theeffects of absorption on LIF are negligible in these thin sections.

4) These valleys can be nearly eliminated by soaking an in vitro bulkaorta sample in saline before collecting the spectra (FIGS. 9a and 9b).This soaking removes most of the Hb, which can freely diffuse from thetissue.

The physical basis for these valleys is quite simple: a portion of theincident excitation light as well as the emitted fluorescence isreabsorbed by the Hb, causing a reduction in the fluorescence intensity.This can be clearly seen from the single layer tissue fluorescencemodel, in which the observed fluorescence intensity S(λ_(x),λm) can bewritten ##EQU1## where F(λ_(x),λm) is the "intrinsic" tissuefluorescence, i.e. the tissue fluorescence from a thin section, free ofabsorption effects; x_(Hb) A_(Hb) (λ_(x),λm)=μ_(Hb) (λ_(x)) +μ_(Hb)(λ_(m)), where A_(Hb) represents the attenuation spectrum of Hb (andsimilarly for SP, where SP refers to fixed tissue absorbers). A_(Hb)(λ_(x),λm) can be thought of as the normalized attenuation matrix of Hb,with x_(Hb) representing its concentration; it can be generated from theHb absorption spectrum if effects of scattering are neglected. A contourmap of A_(Hb) (λ_(x),λm), which clearly shows the Soret absorptionbands, is shown in FIG. 10.

Equation (1) can be rewritten ##EQU2## where S₀ (λ_(x),λm) is thefluorescence observed in the absence of Hb(x_(Hb=) 0). By making thecrude assumption that A_(SP) is a constant (not dependent on wavelength)over the entire EEM, by using values of x_(Hb) and x_(SP) A_(SP)determined with 476 nm excitation, and by using the calculated Hbabsorption matrix, the effect of Hb absorption can be reproduced fromthe soaked aorta EEM (x_(Hb=) 0) by "dividing" Hb absorption matrix intoit in the manner specified by Eq. 2. The result is displayed in FIGS.11a and 11b. The structure of the aorta EEM with Hb (before soaking) isrecovered, although peak intensities, especially in the ultraviolet, aresmaller than in the actual EEM.

Using this method, Hb absorption effects can also be produced in an exvivo aorta sample. The EEM of the ex vivo sample which has presumablynot been exposed to free Hb, is quite similar to that of the soakedaorta. By dividing the Hb absorption matrix into it in the manner of Eq.2, an EEM very similar to that of Hb-containing in vitro aorta isproduced.

This method of correcting for the presence of absorbers within thetissue being examined can be applied to other absorbing components. Theremoval of the effects of absorption can assist in providing a clearerspectrum in which all of the avisibile information regarding tissuefluorescence can be recovered.

The system also assists in identifying the chromophores which contributeto arterial tissue fluorescence spectra. Contour maps provide veryuseful insight about the number and nature of tissue chromophores. Toseparate contributions from different chromophores, maps can be obtainedfrom thin tissue sections, which contain a limited number ofchromophores (e.g. ceroid in necrotic core) and are free of reabsorptioneffects. Also maps of pure compounds, which are suspected tissuechromophores, can be constructed for comparison with tissue maps. Forexample, FIG. 12 contains a map of powdered type 1 bovine collagen.Other components studied include chromophores or structures containingchromophores such as elastin, tryptophan, hemoglobin and flavoprotein.

Although type 1 collagen has emission in the same region as does normalaorta, there are several important differences. Some of these are due toreabsorption of hemoglobin; the collagen map does not show valleys at420, 540 and 580 nm. However, other differences are not related toreabsorption, and indicate that tissue fluorescence in this regioncontains contributions from chromophores other than those present intype 1 collagen.

The methods of the present invention have been used to differentiatenormal and pathologic tissues in human colon and urinary bladder withexcitation wavelengths ranging from 250-500 nm, utilizing fluorescencecontour maps. This contour mapping serves to identify optimal excitationwavelengths for differentiating normal and pathologic tissuefluorescence spectra and to identify tissue chromophores contributing tothe fluorescence of normal and pathologic tissues.

Fluorescence contour maps were collected from 18 colon specimens and 15urinary bladder specimens. Fluorescence contour maps were constructedfrom a series of fluorescence emission spectra recorded using aspectrofluorimeter. Excitation wavelengths were varied in 10 nm stepsfrom 250 to 500 nm. Fluorescence was collected at 5 nm intervals fromλexc+10 nm to 2λexc-10 nm. Incident beam size was ˜2×3 mm, smaller thanthe surface area of all tissue samples, thus absolute intensityinformation has been preserved in the analysis of this data. To correctfor day to day variations in the spectrofluorimeter alignment, all datahas been divided by a fluorescence intensity standard which is run eachday. Emission gratings were blazed at 250 nm. All data presented herehas been corrected for the non-uniform spectral response of thecollection system.

The following method of data analysis was employed for both colon andbladder tissues. An average normal contour map N_(A) (λ_(x),λm), and anaverage pathologic contour map P_(A) (λ_(x),λm) were calculated and wereplotted on a log scale with contours ranging from 50 to 0.5. To comparecontour maps of normal and pathologic tissues, a ratio of the averagecontour maps was constructed as: ##EQU3## and was plotted on a linearscale with contours varying from 2.0 to 0.2. Here, a contour at 1.0indicates regions in which the fluorescence emission from normal andpathologic tissues is the same, while contours at values greater (less)than 1.0 indicate regions where the emission from pathologic tissue isgreater (less) than that from normal tissues Regions of greatestdifference in the fluorescence spectra of normal and pathologic tissueswere assessed from this average ratio map. This information was used indefining a process for determination of tissue type.

To assess how the variability of individual tissues affected thiscomparison, the following ratio maps were constructed for eachindividual normal and pathologic sample: ##EQU4## Data from theseindividual ratio maps was used to test processes suggested in the abovedata analysis procedure.

FIGS. 13 and 14 show average fluorescence contour maps of normal andadenomatous human colon tissue. In the normal tissue fluorescencecontour map, several major fluorescence and absorption bands can berecognized, as summarized in Table 1. Similarly, Table 2 lists the majorabsorption and fluorescence bands found in the adenomatous fluorescencecontour map.

                  TABLE 1                                                         ______________________________________                                        Average Normal Colon Tissue                                                               Fluorescence                                                      (λx, λm)                                                                    Intensity    Chromophore                                          ______________________________________                                        (285, 340)  50.0         Tryptophan                                           (330, 385)  5.0          Chromophore                                                                   Within Collagen                                      (315, 430)  2.5          4-Pyridoxic Acid                                     (345, 465)  5.0          NAD(P)H                                              (460, 520)  4.5          Flavin                                               (λ, 420) (420, λ)                                                                        Hemoglobin (Heme)                                    (λ, 540) (λ, 580)                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Average Adenomatous Colon Tissue                                                          Fluorescence                                                      (λx, λm)                                                                    Intensity    Chromophore                                          ______________________________________                                        (285, 340)  50.0         Tryptophan                                           (340, 470)  2.5          NAD(P)H                                              (460, 520)  2.5          Flavin                                               (λ,420) (420, λ)                                                                         Hemoglobin (Heme)                                    (λ, 540) (λ, 580)                                               ______________________________________                                    

A comparison of these two tables points out several of the interestingdifferences in the fluorescence contour maps of normal and adenomatoustissues Both tissues exhibit tryptophan fluorescence. Qualitatively, theshape and peak fluorescence intensity of this band is very similar forboth types of tissue Two additional fluorescence bands appear to bepresent in the normal tissue fluorescence contour map at (330,385) and(315,430). In the visible region of the spectrum, the fluorescenceintensity of normal tissues is approximately 2× that of adenomatoustissues. In particular, both tissues show a band near (340,470); thisband is 2× higher in normal tissue. In addition, it is more peaked, andslightly shifted in normal tissue relative to adenomatous tissue. Bothtissues show a peak at (460,520), which is 1.8× more intense in normaltissue.

These differences are highlighted in FIG. 15, which shows the ratio ofthe two average contour maps, with contours drawn linearly from 2.0 to0.2. Greater detail is shown in FIG. 16, with contours drawn from 1.0 to0.1. Again contours at 1.0 indicate regions where normal and adenomatoustissues exhibit equal fluorescence intensities, while contours greater(less) than 1.0 indicate regions where adenomatous tissue fluorescenceis greater (less) than normal tissue fluorescence. In the region oftryptophan fluorescence, a contour with a value of 1.0 is shown,indicating the similarity of tryptophan fluorescence in normal andadenomatous tissues. A valley is present at (335,385) with a contourlevel of 0.3 indicating the additional fluorescence band present innormal tissue at this location. A valley is present at (390,430) with acontour of 0.8. Additional valleys are present at (400,495), (440,480),(430,600) and (430,670). These are summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                        Average Ratio Map, Colon Tissue                                               (λx, λm)                                                                  .sup.R AVG.sup.(λ x, .sup.λ m.sup.)                                            Chromophore                                          ______________________________________                                        (330, 385)                                                                              0.3            Chromophore                                                                   Within Collagen                                      (390, 430)                                                                              0.8            Pyridoxic Acid                                                                Lactone                                              (400, 495)                                                                              0.4            ?                                                    (440, 480)                                                                              0.4            ?                                                    (430, 600)                                                                              0.7            ?                                                    (430, 670)                                                                              0.8            ?                                                    ______________________________________                                    

The values of the individual ratio maps at these locations of(λ_(x),λm), can be divided by the intensity of the ratio map at(290,340). Two of these values, at (330,385) and (440,480),independently provided useful diagnostics for differentiating normal andadenomatous tissues (FIGS. 17 and 18). In addition, a binary schemeutilizing information at (390,430) and (330,385) provided an accuratediagnostic process (FIG. 19).

FIGS. 20 and 21 show average fluorescence contour maps of normal bladderwall and tumor. Major fluorescence and absorption bands are summaried inTables 4 and 5. Again, in the region of tryptophan fluorescence bothnormal and tumor tissues have similar emission. Although both tissuesshow a fluorescence band at (325,385), it is 3× stronger in normaltissue. Both tissues show fluorescence peaks at (350,470) with normaltissues exhibiting 2× as much fluorescence intensity. Also at (470,520)the fluorescence of normal tissue is 2× as high as that of tumor tissue.The average tumor map exhibits a unique peak at (315,430).

                  TABLE 4                                                         ______________________________________                                        Average Normal Bladder Tissue                                                             Fluorescence                                                      (λx, λm)                                                                    Intensity    Chromophore                                          ______________________________________                                        (285, 340)  50.0         Tryptophan                                           (325, 385)  15.0         Chromophore                                                                   Within Collagen                                      (350, 470)  10.0         NAD(P)H                                              (470, 520)  10.0         Flavin                                               (λ, 420) (420, λ)                                                                        Hemoglobin (Heme)                                    (λ, 540) (λ, 580)                                               ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Average Bladder Tumor                                                                     Fluorescence                                                      (λx, λm)                                                                    Intensity    Chromophore                                          ______________________________________                                        (285, 340)  50.0         Tryptophan                                           (330, 385)  5.0          Chromophore                                                                   Within Collagen                                      (315, 430)  3.0          4-Pyridoxic Acid                                     (350, 470)  5.0          NAD(P)H                                              (460, 515)  5.0          Flavin                                               (λ, 420) (420, λ)                                                                        Hemoglobin (Heme)                                    (λ, 540) (λ, 580)                                               ______________________________________                                    

These differences are highlighted in FIGS. 22 and 23 which show theratio contour map of tumor and normal bladder tissue for two sets ofcontours (2.0-0.2, 1.0-0.1). Table 6 summarizes the valleys present inthis ratio map. Valleys are present at (330,385), (370,435), (400,495),(450,485), (420,640), and (390,670). Values of the individual ratio mapsat these locations can be divided by the value of the tryptophan peak(290,340). FIG. 24 shows an example of a diagnostic procedure which canbe defined from this data.

                  TABLE 6                                                         ______________________________________                                        Average Ratio Map, Bladder Tissue                                             (λx, λm)                                                                  .sup.R AVG.sup.(λ x, .sup.λ m.sup.)                                            Chromophore                                          ______________________________________                                        (330, 385)                                                                              0.4            Chromophore                                                                   Within Collagen                                      (370, 435)                                                                              0.8            Pyridoxic Acid                                                                Lactone                                              (400, 495)                                                                              0.5            ?                                                    (450, 485)                                                                              0.4            ?                                                    (420, 640)                                                                              0.9            ?                                                    (390, 670)                                                                              0.6            ?                                                    ______________________________________                                    

Because of the voluminous information in the 2-D plots, a model isuseful in interpreting the data. Even if the types and concentrations ofall chromophores were known, quenching, spectral shifts, and energytransfer due to the local environment would alter a calculated spectrum.But, in spite of these many unknowns, a "landscape" model of chromophorebehavior does provide some general insights for locating distinctivefeatures for comparative spectral diagnosis.

The model begins with the reasonable assumption that tissue contains afew relatively strongly fluorescing chromophores, a background of weakerones, and absorbers which are probably non-fluorescent. The strongchromophores, with relatively high quantum yields or concentrations (orboth), will generate "peaks" on the contour plot. In addition, thesechromophores generally have a broad base which tends toward shorterwavelengths for excitation; and due to the general anharmonicity ofhigher vibrational levels, it tends toward longer wavelengths foremission. Combining with the weaker chromophores, this forms an averageelevation of signal background from which the peaks arise. The absorberscut vertically and horizontally across the spectral terrain. The percentdecrease along the line should be approximately constant to the extentthat the penetration depth is constant. This is distinguishable from a"pass" between two spectral peaks, which is just lesser fluorescence,not absorption. Thin sections will be quite helpful in identifyingabsorption features by their absence.

Distinguishing diseased vs. healthy tissue is essentially distinguishingone set of chromophore signals from another. If two tissues havedifferent amounts of one chromophore, then the corresponding absolutesignals might be measured; however, this is dependent on collectiongeometry. Comparisons, or ratios, are less geometry dependent, butappropriate positions on the 2-D surface need to be selected. The datais normalized so as to again avoid geometry-dependent ratios.

The spectral landscape model has three regions: 1) fluorescent peaks, 2)an average elevation, or plateau, and 3) absorption valleys. Of these,region 1 is the most appealing as a discriminant for tissue type, as itwould show a characteristic chromophore. Region 3 is susceptible tovariability or leaching as has been shown for the case of heme, andregion 2, being comprised of fluorescence from several chromophores, islikely to have the least variation overall. Signal change due to achange in one of the chromophores would be diminished by the fraction ofthe total signal contributed by that chromophore. In other words,instead of comparing absolute peak heights, they should first benormalized relative to the average elevation. Ideally, normalizationwould be relative to selected regions thought to be "average"; apractical compromise is the average height from the total integratedsignal of the entire plot. Peaks then have signals >1, and valleysgenerally <1. The comparison signal Rλ for each set of excitation andemission wavelengths, equals the diseased signal Dλ, divided by thenormal tissue signal, Nλ.

    Rλ=Dλ/Nλ

If subtraction is used instead of the ratio, then this number should benormalized to the signal strength at that wavelength to indicate thefractional, rather than absolute, change:

    Sλ=ΔDλ/Nλ,

where ΔDλ=Dλ-Nλ and therefore Sλ=Rλ-1.

This gives the normalized differential signal Sλ. N is in thedenominator, since it is desirable to determine the deviation of thediseased tissue from normal. When the "peak" value is selected, itshould be integrated over the top 10%-20% of the peak, therebyincorporating a substantial volume of the fluorescence signal;otherwise, data is discarded, and the noise level is increased.

An alternative approach is to normalize two contour plots to the samechromophore peak, then ratio them. This would compare amplitudes ofother peaks to the first. However, the ratioed contour plot is dependenton which peak is selected for normalization, and the information fromthe rest of the plot, which provides the total integrated signal, is ineffect, unused.

Additional information is contained in the peak shape. If the differentenvironments of the tissue cause spectral shifts, then this may beemphasized by ratioing normalized peaks on two contour plots. In thiscase a smaller integrated area may be better for normalization.Deviations from unity will indicate different slopes or shapes.

To avoid overemphasizing weaker parts of the spectrum, a weightingfactor may be introduced: Assuming that the signal to noise ratio variesas the square root of the signal, the ratio signal should be multipliedby the square root of Nλ to yield Wλ, the weighted normalized ratiocontour plot comparing the two tissue types.

    Wλ=NλRλ.

These computations should be straightforward for numerical peakcomparison. Large differences in regions where the fluorescence is weakwill be de-emphasized.

We claim:
 1. A method of processing spectra generated from the inducedfluorescence of tissue comprising:irradiating a portion of tissue over arange of periodically separated excitation wavelengths with radiationfrom a light source to induce autofluorescence of the tissue such thatthe tissue emits fluorescent radiation at a multiplicity of emissionwavelengths; forming a spectrum of the intensity of the emittedradiation from the recorded fluorescing radiation at each of themultiplicity of excitation and emission wavelengths; repeating theirradiating and forming steps to provide a plurality of spectra;averaging the plurality of spectra to produce an average spectrum; andidentifying fluorophores present in the tissue that have predeterminedfluorescence characteristics which correlate with spectral features inthe average spectrum, the feature having identified excitation andemission wavelengths present in the average spectrum.
 2. The method ofprocessing spectra of claim 1 wherein the tissue comprises arterialtissue.
 3. The method of processing spectra of claim 1 wherein thetissue comprises colon tissue.
 4. The method of processing spectra ofclaim 1 wherein the tissue comprises bladder tissue.
 5. The method ofprocessing spectra of claim 1 further comprising the step of forming adifference spectra by taking the difference between the formed spectrumand a reference spectrum.
 6. The method of processing spectra of claim 1further comprising identifying a region within the spectrum wherein theintensity in the region is reduced by a component of tissue whichabsorbs radiation.
 7. The method of processing spectra of claim 6further comprising adjusting the intensity in the region to correct fora contribution to the spectrum by the absorbing component of tissue. 8.The method of processing spectra of claim 1 further comprising the stepof forming a ratio spectrum by dividing the formed spectrum by areference spectrum.
 9. A method of diagnosing bodily tissuecomprising:inserting a catheter containing an optical fiber into a bodylumen and positioning a distal end of the catheter adjacent to tissue tobe diagnosed; coupling a proximal end of the catheter to a radiationsource such that the radiation is transmitted along the optical fiberand directed onto the tissue to be diagnosed; inducting autofluorescencewith the tissue with the radiation source with a multiplicity ofperiodically separated excitation wavelengths; recording theautofluorescent radiation emitted by the tissue at a multiplicity ofemission wavelengths; forming a spectral matrix of the intensity of theemitted radiation from the recorded radiation at each of themultiplicity of excitation and emission wavelengths; repeating theinducing and recording steps to provide a plurality of the spectralmatrices; averaging the plurality of spectral matrix to produce anaverage spectral matrices; and identifying fluorophores present in thetissue from the average spectral matrix to diagnose a condition of thetissue.
 10. The method of claim 9 further comprising comparing thespectral matrix with a reference matrix to determine the presence ofabnormal tissue.
 11. The method of claim 9 wherein the radiation sourcecomprises a laser.
 12. The method of claim 9 wherein the lumen comprisesan artery such that fluorophores of atherosclerotic material within theartery are identified.
 13. The method of claim 9 further comprisingidentifying a portion of the spectral matrix wherein the intensity ofthe emitted radiation is reduced by a component of tissue which absorbsradiation.
 14. The method of claim 13 further comprising adjusting theintensity of the identified portion of the spectral matrix to correctfor the reduced intensity.
 15. A method of diagnosing vascular tissuecomprising:inserting a catheter containing an optical fiber into avascular lumen and positioning a distal end of the catheter adjacent tovascular tissue to be diagnosed; coupling a proximal end of the catheterto a laser radiation source such that the laser radiation is transmittedalong the optical fiber and directed onto the tissue to be diagnosed;inducing inelastically scattered light to be emitted from the vasculartissue with the laser radiation with a multiplicity of periodicallyseparated excitation wavelengths; recording the scattered radiationreturning from the tissue at a multiplicity of emission wavelengths;forming a spectral matrix of the intensity of the emitted radiation fromthe recorded radiation at each of the multiplicity of excitation andemission wavelengths; repeating the inducing and recording steps toprovide a plurality of spectral matrices; averaging the spectralmatrices to provide an average spectral matrix; and identifying abnormaltissue present in the tissue from the average spectral matrix.
 16. Themethod of claim 15 further comprising comparing the spectral matrix witha reference matrix to determine the presence of abnormal tissue.
 17. Themethod of claim 15 wherein the lumen comprises an artery such thatfluorophores of atherosclerotic material within the artery areidentified.
 18. The method of claim 15 further comprising identifying aportion of the spectral matrix wherein the intensity of the emittedradiation is reduced by a component of tissue which absorbs radiation.19. The method of claim 18 further comprising adjusting the intensity ofthe identified portion of the spectral matrix to correct for the reducedintensity.
 20. The method of claim 15 wherein the inelasticallyscattered light comprises fluorescence.